We have purified the muscarinic cholinergic receptor from bovine brain to near homogeneity, and have studied the binding of pirenzepine to this receptor at various stages of purification. In accord with previously published work, we found that in membrane-bound receptors, pirenzepine binds preferentially to receptors from cortex, compared to receptors from the pons/medulla. After digitonin solubilization, however, this difference is no longer detectable. We have identified and characterized an endogenous muscarinic factor from acid-extracts of brain. This factor inhibits ligand binding to the muscarinic receptor and acts like an agonist in inhibiting prostaglandin-stimulated adenylate cyclase in NG108-15 neuroblastoma-glioma cells. We found that the calcium channel inhibitor verapamil afects the binding characteristics of muscarinic receptors. These effects were studied in detail.